EP1292671B1 - Method for obtaining characterised muscle-derived cell populations and uses - Google Patents

Abstract

A method for obtaining cell populations derived from the muscular tissue and their use for preparing cell therapy products includes culturing cells previously removed by biopsy from skeletal muscular tissues, identifying the different types of cells present at different stages of culture, selecting the culture stage on the basis of the required cell population and collecting the selected culture stage for preparing a cell therapy product. The invention also concerns cell populations derived from muscular tissue obtained by implementing the method whereof the dominant cell type is CD34+, CD15+ or CD56+ or Class 1+HLA, or comprises a doubly negative CD56−/CD15− cell type or may comprise more minority CD10+, Stro-1+ and CD 117+ cell types.

Description

Cell therapy is a technique with high potential for the treatment of many pathologies. The principle of cell therapy is based on the possibility of reconstituting damaged tissue or restoring lost or altered tissue function within a tissue, from specific cells cultured ex vivo and transplanted to the diseased tissue. Another advantage of cell therapy is the possibility of using the transplanted cells as a delivery platform for a biologically active product, if necessary after genetic modification of the cells before transplantation. Many cell therapy trials are described from primary cultures of different cell types. These include neuronal cell transplants performed for the treatment of Huntington's disease (1) or Parkinson's disease (2), Langerhan's islet cell transplants performed for the treatment of diabetes (3), or Myoblastic cell transplants performed for the treatment of Duchenne muscular dystrophy (4, 5, 6, 7) or after genetic modification of the cells, for the treatment of dwarfism (8), hemophilia (9) and Parkinson's disease (10).

The regeneration of skeletal muscle is ensured by the satellite cells which are myogenic myonuclear cells situated under the basal lamina of the muscular fibers. Following an injury, these cells leave the quiescent state to enter an active proliferation phase and take the name of myoblasts. Subsequently, the myoblasts fuse to form the myotubes. Myoblast transplantation trials have been conducted in humans to treat Duchenne muscular dystrophy and Becker muscular dystrophy (4, 6, 7, 11). Although the functional effect of transplants described in these studies remains limited, no side effects have been reported in terms of infection or carcinogenesis.

Moreover, the use of myoblastic cells in the treatment of heart disease, and in particular of post-ischemic heart failure has been considered. Indeed, unlike muscle tissue, myocardial tissues lack stem cells that can form cadiomyocytes and regenerate tissues. The most radical treatment of post-ischemic heart failure remains at present, heart transplantation. However, the scarcity of grafts limits this therapeutic use. Transplantation of muscle tissue cells into cardiac muscle has therefore been considered as an alternative to cardiac transplantation. Myoblast transplantation studies in infarcted myocardial tissue have been performed in rats, rabbits or dogs (12, 13, 14). The results of these studies have shown the feasibility and some functional efficacy of such transplants. Fetal cardiomyocyte transplant assays in a model of iatrogenic heart failure in mice also showed some functional benefit. However, the use of fetal cells poses, for the clinical application, many ethical, immunological and cellular supply problems. The use of a population of myoblastic cells derived from muscle Skeletal is therefore a particularly interesting alternative for the preparation of cell therapy product for the treatment of post-ischemic heart failure, or even for the treatment of heart diseases of various origins.

One of the most important cell types in muscle tissue is the satellite cell, a precursor of the myoblast. This is the cell type that has been used in various clinical studies. However, muscle tissue contains other cell types. In particular, certain cells of muscular origin could also be used for the reconstitution of a hematopoietic potential (15, 16). An in vitro study has also demonstrated the presence in human muscle of progenitors that can differentiate in the long term into cartilaginous or bone tissue (17). Examples of media allowing differentiation into adipose, cartilage or bone tissue are described for mesenchymal stem cells (22).

Consequently, taking into account the differentiation properties of cells of muscular origin, the use of these cells in cell therapy appears interesting for the treatment of numerous lesions affecting the tissues of the hematological and immunological system, the bone, adipose, cartilaginous tissues, muscle or vascular.

One of the major difficulties of cell therapy remains obtaining a sufficiently large, homogeneous cell population whose degree of differentiation is adapted to the desired effect.

• une étape de prélèvement des tissus musculaires par biopsie,
• une étape d'éminçage,
• une étape de dissociation des fibres musculaires par action enzymatique,
• une étape de séparation des cellules individualisées par filtration,
• une étape de sélection des cellules myoblastiques par clonage ou par tri cellulaire.

Myoblastic cell preparation methods and their use in cell therapy are described in the state of the art (4, 18, 19, 20, 21, 25, 26, 27). The majority of processes include:

• a step of collecting muscle tissue by biopsy,
• a slicing step,
• a step of dissociation of the muscle fibers by enzymatic action,
• a step of separating the individualized cells by filtration,
• a step of selecting myoblastic cells by cloning or by cell sorting.

In order to obtain a sufficiently dense and rich population, or even pure populations of myoblastic cells, it has been proposed to select myoblastic cells on the basis of the expression of specific markers. Thus, the selection of myoblastic cells can be done by cloning the cells and subsequently characterizing the clones obtained by cytofluorimetry and then selecting the skeletal muscle cells expressing the CD56 antigen (7). A direct method of sorting myoblastic cells expressing the CD56 antigen by flow cytofluorometry and its advantages for obtaining a pure culture of myoblasts are also described in the state of the art (21).

The conserved cells are then cultured in a modified culture medium and specially adapted for culturing the myoblasts (23).

The present invention results from the observation that cells derived from skeletal muscle tissue have a potential for regeneration of many tissues depending on their degree of differentiation. The proposed invention therefore makes it possible to provide well-characterized cell populations of muscular origin.

1. a) prélèvement et éminçage d'une biopsie musculaire ,
2. b) dissociation enzymatique des fibres et des cellules musculaires et séparation des cellules individualisées par filtration,
3. c) mise en culture des cellules d'origine musculaire ainsi obtenues dans un réacteur de culture de cellules adhérentes en présence d'un milieu comprenant MCDB 120 ET D-valine substituée à la L-valine suivie le cas échéant d'une ou plusieurs phases d'expansion,
4. d) identification des types cellulaires présents aux différents stades de la culture par l'analyse de marqueurs cellulaires spécifiques,
5. e) choix du stade de culture pendant lequel le type cellulaire recherché est en proportion dominante dans la population de cellules,
6. f) récolte d'une population de cellules au stade de culture choisi en e),
7. g) le cas échéant, congélation des cellules prélevées à l'étape choisie.

The present invention provides a method of obtaining a cell population comprising a dominant cell type, from a muscle tissue biopsy, said method comprising the following steps:

1. a) sampling and cutting of a muscle biopsy,
2. b) enzymatic dissociation of the fibers and muscle cells and separation of the individualized cells by filtration,
3. c) culturing the cells of muscle origin thus obtained in an adherent cell culture reactor in the presence of a medium comprising MCDB 120 and D-valine substituted for L-valine followed, where appropriate, by one or more phases expansion,
4. d) identification of the cell types present at the different stages of the culture by the analysis of specific cell markers,
5. e) choice of the culture stage during which the desired cell type is in a dominant proportion in the cell population,
6. f) harvesting a cell population at the culture stage selected in e),
7. g) where appropriate, freezing the cells taken at the chosen step.

Step d) is optional insofar as, when the process is used several times under the same conditions, the experimenter knows the cell types present at the different stages of culture and their relative proportions without having to repeat the step of identification.

It has indeed been found that the identification step (d) led to substantially identical results when the same process was repeated.

In a particular embodiment of the invention, the method further comprises the implementation of depletion techniques or enrichment before the culturing step c) or before expansion, in order to modify the proportions of the different cell types.

The terms "cell population" or "cell population" mean any population of non-pure cells, generally containing a dominant cell type and one or more minority cell types. The dominant cell type is the cell type whose proportion in the cell population is the highest. Preferentially, a dominant cell type is the cell type whose proportion in the cell population exceeds 50%. A cell therapy product suitable for human administration comprises an isotonic solution in which the cells are resuspended. This solution must be free of toxic components present in the freezing media. Such a component is, for example, DMSO.

The invention results in particular from the observation that the method makes it possible to obtain a population of cells whose composition is adapted to the desired therapeutic effect. It makes it possible to obtain a population of cells whose dominant cell type expresses the CD56 + marker and the HLA class I marker without prior cloning, nor positive selection of the cells expressing the CD56 + marker.

The muscular biopsy is usually performed by collecting cubes of 2 to 4 cm side. From 0.05 grams to several tens of grams can be taken as needed. One of the advantages of the method is that it makes it possible to obtain a very large number of cells of a cell type in a dominant proportion, varying from a few thousand to several billion, depending on the cell type sought, the number of expansions carried out, and the time allotted for each passage. By way of example, the method makes it possible to produce up to several hundred million cells expressing the CD56 antigen within two to three weeks. The process allows for many phases of expansion, allows the theoretical achievement of at least 100 billion cells expressing CD56 + antigen and HLA class I antigen after 8 to 9 expansions. The muscular tissue taken is a skeletal muscle tissue, preferentially taken from the adult, the young adult, the adolescent or the child. In one embodiment of the invention, the muscle tissue removed is fetal skeletal muscle tissue. The cells can be obtained especially from the vastus externa, vastus interna, biceps, quadriceps, hamstrings, gastrocnemius, peroneal, deltoid, dorsal, sternocleidomastoid, intercostal, homo-hyoid, right or psoas.

The slicing consists in cutting the biopsy into sections of a size preferably less than 0.5 mm placed in a suitable culture medium. Slicing is an essential step to enable effective subsequent enzymatic dissociation. Emining can be done manually using fine scissors. Unexpectedly, however, it has been found that when the slicing step is carried out in an assisted manner, for example using knife crushers powered by electrical or mechanical energy, the method of the invention in which the culture medium is adapted to myoblast differentiation makes it possible to obtain a population whose percentage of cells expressing the CD56 antigen is particularly high. An example of such a usable crusher is the Medimachine ® mill (distributed by Becton-Dickinson).

Therefore, in one embodiment, the method of the invention is characterized in that the slicing step is assisted by mechanical or electric knife mills.

Muscle tissues consist of muscle fibers. The satellite cells are located under the basal lamina of the muscle fibers. The step of dissociation of the muscle fibers and detachment of the satellite cells is therefore a necessary step for their isolation. The dissociation step consists in the use of digestion enzymes of the extracellular matrix, it can be completed by a mechanical dissociation by suction and delivery of the suspension through a pipette.

• toutes les collagénases, incluant les types IA, S et H partiellement purifiées, ainsi que la forme purifiée commercialisée sous le nom de Libérase par Roche-Boehringer,
• les trypsines, de toutes origines, en solution dans des tampons contenant ou non de l'EDTA,
• les dispases (aussi connues sous le nom de protéases),
• la pronase,
• les élastases,
• ou encore, les hyaluronidases.

The choice of the enzymes and their concentrations used for the dissociation of the muscle fibers and the satellite cells of the emine is guided by the study of their enzymatic efficiency, the desired criteria are a concentration of enzyme as low as possible and a time of minimal incubation for similar efficiency. The yield of cells obtained after filtration depends in part on the quality of the enzymatic dissociation step. Digestion enzymes that can be used in the process of the invention, alone or in combination, are, for example:

• all collagenases, including partially purified IA, S and H types, as well as the purified form sold under the name of Liberase by Roche-Boehringer,
• trypsins, of all origins, in solution in buffers containing or not containing EDTA,
• dispases (also known as proteases),
• the pronase,
• elastases,
• or else, hyaluronidases.

Preferably, the dissociation step is in two stages; a first incubation in the presence of collagenase and a second incubation in the presence of trypsin. When liberase is used, particularly effective levels of use in the emine are between 0.05 to 2 mg / ml. The incubation time at 37 ° C. for such concentrations is then chosen in a time range extending from 15 minutes to 2 hours. Preferably, the activity of dissociation enzymes is neutralized after dissociation or detachment of the cell layer in order to avoid cell damage.

After neutralization of the enzymatic activity, by adding, for example, fetal calf serum, autologous human serum, or allologous human serum from a compatible group, or an enzymatic activity inhibitor, the product digestion is then filtered through sieves, under gravity pressure to remove undissociated fibers and collect the cells detached from the muscle fibers. Preferably, a filtration step will be carried out in two steps: a filtration step on a 100 μm sieve, then a second step on a 40 μm sieve.

The cells collected after filtration are transferred to a culture reactor in the presence of a medium whose composition allows their growth and / or their differentiation. The composition of the medium is chosen according to the dominant cell type desired at the end of culture. At this stage, some of the preparation can be frozen. This can be a part of the initial preparation or a subpopulation from an enrichment or depletion stage. The culture media used contain one or more growth and / or differentiation factors whose role is to direct the progenitor cells to a specific differentiation pathway and to proliferate them. By way of example of growth factors, mention may be made of fibroblast growth factors, bFGF, aFGF, FGF6, hepatocyte growth factor, HGF / SF, epidermal factor, EGF and the various factors characterized such as IGFs. -1, PDGF, LIF, VEGF, SCF, TGFb, TNFa, IL-6, IL-15, NGF, neuregulin, thrombopoietin and growth hormone. They can be associated with different hormones or active molecules that can be used in the composition of media, such as glucocorticoids (natural or semi-synthetic, ie hydrocortisone, dexamethasone, prednisolone or triamcinolone), progestagens and derivatives ( progesterone), estrogens and derivatives (estradiol), androgens and derivatives (testosterone), mineralocarticoids and derivatives (aldosterone), LH hormones, LH-RH, FSH and TSH, thyroid hormones T3, T4, retinoic acid and its derivatives, calcitonin, prostaglandins E2 and F2 / alpha or parathyroid hormone.

Prior to culturing, or during an expansion phase, the preparation may be enriched or depleted. These operations are carried out by those skilled in the art using the various techniques proposed in the state of the art to operate a selective sorting. These techniques rely on the identification of extracellular antigens characteristic of this or that cell type by specific reagents. By way of example, CD34 and CD56 antigens expressed on certain cells present within the muscle cell population may be used. Initial sorting of the total cell population according to the expression of these two antigens thus makes it possible to separate two groups. In particular, it makes it possible to separate CD34 + cell types from CD34 cell types. It has been shown that the CD34 + population generates a dominant CD15 + cell type, CD56- not expressing desmin. The CD34 population generates a dominant CD56 +, CD15-expressing desmin cell type. In rare cases, the population CD34- generates a population CD56-, CD15-. In particular, the method of the invention comprises a stage of depletion of CD34- or CD34 + cells leading respectively to a population comprising a dominant cell type C015 + or CD56 +.

One embodiment of the method of the invention therefore relates to a method for obtaining a population of cells of which a dominant cell type is the myoblastic cell type, characterized in that it comprises a stage of depletion of CD34 + cells before the cell culture or during one of the expansion phases.

The cells are then cultured in a reactor suitable for the culture of adherent cells. In order to overcome the constraints of controlling the stirring speed, its regularity and the homogeneity of the preparations, the culture reactor is preferably static. It must have a large culture surface compared to conventional carriers (petri dishes, flasks) so as to harvest in a few days a large cell population. An example of such a culture reactor is the tray culture device (single, double and / or multi-stage).

The culture device used in the method also allows sterile sampling of the cells in order to carry out the samples necessary for the identification of the cell types present at the different stages of the culture by analysis of specific markers. It allows the emptying of media, the washing and detachment of cells and finally their harvest in a sterile manner.

In a preferred manner, bags are used and specially adapted sterile tubings connect the bags to the reactor to allow transfer of media or cell harvesting. This device thus makes it possible to perform a large number of operations in a closed system. Depending on the desired cell population, the number of days of culture varies from 0 to 45 days.

In addition, the culture can be continued by conventional expansion or perfusion techniques for a period of up to several months.

In order to increase the number of cells harvested, one or more expansion phases are possible. The expansion phases comprise a step of detaching the cells, washing the cells and restoring culture to a larger culture surface, the solutions and enzymes used to carry out these steps being well known to those skilled in the art. In particular, in a preferred embodiment using a culture medium suitable for myoblast differentiation, the method of the invention comprises at least three phases of cell expansions. Such a method makes it possible to multiply the number of cells without substantially modifying the proportions of the cell types obtained at the end of culture of each expansion.

Kinetics of differentiation is carried out in the method of the invention by identifying cell types present in the cell populations obtained at different stages of the culture. In the text that follows, we will designate the step of identifying the cell types present at different stages of the culture by the term "characterization of cell populations". This characterization is carried out from samples taken during the cultivation, during the culture and during the harvesting of the cells. The characterization of the cell populations can also relate to a cell culture carried out in parallel on a smaller scale but under identical or equivalent conditions in terms of culture medium and method of carrying out the expansion phases. The characterization relates to the adherent cells or the cells present in the supernatant. The different stages of the culture are counted in days starting from the day of culture in a reactor until the appearance of the first differentiated cells or obtaining a degree of confluence of the cells of at least 20 to 50%. . The function of cell population characterization is to identify all cell types and their proportions according to the stage of culture. In particular, it makes it possible to highlight the dominant cell types according to the stage of the culture. The method of the invention thus provides a means of selecting a cell population according to the characteristics of the different cell types present in the population.

This characterization is carried out by the analysis of cell markers by flow cytofluorometry or FACS, after labeling the surface antigens or any specific antigen of the different cell types to be analyzed. In the present text, the term "cellular markers" denotes any cell antigen that can provide information alone or in combination with other markers on a cell type. Any type of cell marker can be used by the method, the choice of markers used will be guided by the choice of cell types that one wishes to characterize.

Any technique capable of characterizing a cellular antigen can be implemented in the method of the invention. By way of example, Western Blot or immunocytochemistry may be mentioned without being limiting. The techniques allowing the characterization of messenger RNAs encoding said antigens can be used in an equivalent manner.

The cellular markers analyzed for the identification of cell types are specifically chosen from the following markers: CD10, CD13, CD15, CD16, CD34, CD38, CD40, CD44, CD45, CD56, CD71, CD80, CD117 or the following structures, CD138, HLA-Class I, HLA-class II, VLA3, VLA5, VLA6, ICAM-1, VCAM and desmin. In a preferred manner, the markers CD10, CD13, CD15, CD34, CD44, CD56, CD117 and HLA Class I and desmin are used. These markers are identified by the use of specific monoclonal antibodies. Table 1, hereinafter provided in the experimental part, indicates, for each of the markers, the cell types which conventionally express the corresponding antigens. It should be noted that these cell markers were initially developed for the characterization of the immuno-hematological system. An originality of the invention consists in the use of these markers for the characterization of cells of muscular origin at the different stages of culture or expansion.

The method of the invention is therefore implemented through the analysis of cellular markers not conventionally used in the characterization of cells derived from muscle tissue. These cell markers are, for example, CD10, CD13, CD15, CD34, CD44, CD45, CD117 and HLA Class I.

It has indeed been observed that the implementation of the method of the invention allows the demonstration of an evolution over time of the dominant cell type present in the culture. Surprisingly, in the early stages of culturing muscle tissue cells and in a medium suitable for myoblastic differentiation, the majority populations are those expressing markers of marrow progenitor cells and cells of the lymphoblastoid system. More precisely, a majority of CD34 + / 45- phenotype cells were observed on day 0 and day 1, as well as the presence of a CD117 + minority cell type in the non-adherent cell population. The evolution is then marked by an increasing increase in the proportion of CD15 + and / or CD56 + cells, and a decrease in CD34 + populations. Nevertheless, the dominant cell type remains CD34 + from D0 to D5. A gradual transition from a dominant cell type CD34 + to CD56 + cell is observed. On the other hand, the proportion of CD13 +, CD44 +, CD71 + cell types increases with time. Minority populations CD138 +, HLA-Class II + and CD38 + are also observed. At the end of the culture, the dominant cell type is of CD56 + phenotype, especially from the J5 stage until the end of the culture. CD56 + cells also express CD10, CD13, CD44, desmin, HLA-class I, CD44, VLA-3, VLA-5 and VLA-6. These markers characterize myoblastic cells. A second predominant cell type consists of cells expressing CD15, CD10, CD13 and HLA-Class I. A population CD56- / CD15- / CD13 + is also present in variable proportions constituting a third predominant cell type. In this population, one part expresses the desmin and one part does not express it. The implementation of the method of the invention has thus made it possible to distinguish three cell types present in the cell populations, the respective proportions of which change significantly during the course of the culture, the CD34 + cells, the CD15 + cells and the CD56 + cells. .

In a preferred embodiment of the method of the invention, the characterization of the cell populations consists in determining the respective percentage of the three CD34 +, CD15 + and CD56 + cell types at the different stages of the culture.

In one embodiment of the invention, a desired cell type in a cell population is separated after selection of the culture stage during which the desired cell type is in the desired qualitative or quantitative state, particularly after selection of the culture during which the desired cell type is dominant. The presence in predominant proportion of the cells to be separated facilitates their preparation. The invention thus provides a method for obtaining a cell population of high purity. By high purity cell population is meant a population of cells in which the dominant cell type is in excess of 50% in the cell population. It is clear that one skilled in the art can implement the various techniques proposed in the state of the art for a selective sorting of said cells. By way of example, mention may be made of cloning, flow cytofluorometry or immunoaffinity or immunomagnetic columns using antibodies specific for the cells in question.

In another embodiment, on the contrary, the method of the invention is characterized in that it does not include purification steps, positive selection, or cloning of a specific cell type. By positive selection, it is necessary to understand any step for sorting the cells on the basis of at least one particular characteristic and in particular the expression of a cellular marker. It has been shown in particular that the method of the invention, unlike the methods described in the state of the art, makes it possible to obtain a cell therapy product comprising a dominant myoblast-type cell type, without a preferential selection step. of the myoblastic cell type. The absence of cloning steps, purification or positive selection, significantly improves the final yield obtained after the expansion procedures in terms of numbers of cells obtained.

In the method of the invention, after stopping the culture at the selected culture stage, an aliquot of cells can be removed and cultured separately. The medium may be the same or of different composition. The culture media are selected according to the desired differentiation pathway. An example of a medium for differentiation into myoblasts, endothelial cells, smooth muscle cells or myofibroblasts is the MCDB 120 medium supplemented with fetal calf serum (23) which in particular comprises a glucocorticoid such as dexamethasone and bFGF. Another example of a preferred medium is the modified MCDB 120 medium substituting L-valine for D-valine. It has indeed been found that such a modification makes it possible to obtain a particularly selective medium for the differentiation of the cells into myoblasts. Such a medium makes it possible to obtain, with the method of the invention, a population comprising a very large number of cells, most of which are of the CD56 + or HLA Class 1 type.

A differentiation medium in adipose tissue contains, in particular, dexamethasone, isobutylmethylxanthine and, if appropriate, serum fetal calf, indomethacin and insulin. Maintaining differentiation is achieved in medium containing 10% fetal calf serum and insulin. To obtain a differentiation in cartilage tissue, the cells are centrifuged in micromass and cultured in medium without serum but containing TGF-beta3. A bone tissue differentiation medium contains dexamethasone, beta-glycerol phosphate, ascorbate, and, where appropriate, fetal calf serum.

The cells are harvested at the selected culture stage depending on the cell population that is desired. Non-adherent cells present in the supernatant and / or adherent cells can be harvested by this method. The cell populations present in the supernatant and adherent cells may be of different composition. Consequently, the mode of harvesting the cells will be a function of the desired cell population. The harvesting of the adherent cells is carried out by enzymatic dissociation of the cells and detachment of the cell layer by techniques known to those skilled in the art. The non-adherent cells are harvested by aspiration.

The implementation of the above method including the establishment of kinetics of differentiation from cells derived from muscle tissue biopsies allows obtaining characterized cell populations.

The cell populations can be obtained by a method similar to the method for obtaining cell populations described above, but not including a step of identifying the cell types at different levels. cellular stages. Indeed, the step of identifying the cell types is necessary for the choice of the harvesting stage. Since, having carried out at least once the process of the invention, the skilled person knows the optimal stage of harvesting cells according to the desired population, it can obtain the same types of populations by limiting the number of markers used for the characterization of cell populations and the stages at which it performs this characterization. Thus, by "populations of cells that can be obtained", it is therefore necessary to include a population of cells obtained by the process of the invention not necessarily comprising a step of characterizing cell populations within the meaning of the invention, such as as described above. The optimal harvest stage is known by implementing a differentiation kinetics on a prior culture, carried out under the same conditions.

The different cell populations that can be obtained by the method of the invention, with or without an identification step, are of different types depending on the culture stage chosen.

In particular, at the early stages of cell culture, the invention provides a cell population whose dominant cell type expresses the CD34 marker. When only the non-adherent cells are harvested at the early stage of culture, the cell population also includes a minority cell type of CD117 + phenotype.

By carrying out the method described above in which the culture medium is adapted for myoblastic differentiation, the invention provides a cell population whose dominant cell type expresses the CD15 marker.

Finally, by carrying out the method described above and using a culture medium suitable for myoblastic differentiation, a preferred embodiment of the invention provides a cell population whose dominant cell type is myoblastic cells. Myoblastic cells are characterized by the analysis of CD56 marker expression. They are preferably characterized by CD56 marker expression analysis in combination with the analysis of CD10, CD13, CD44, HLA Class I and desmin markers. Analysis of the expression of desmin, an intracellular cytoskeletal protein specific to myoblastic cells and differentiated muscle cells requires a suitable FACS labeling and analysis protocol, detailed in the experimental section. The cell population additionally comprises a doubly negative population CD56 ^- / CD15 ^- .

The invention provides in particular a cell population derived from the same muscle biopsy and comprising from 50 × 10 ⁶ to 800 × 10 ⁹ cells, preferably at least 500 × 10 ⁶ cells, of which at least 50% of the cells, and better still at least 60%, and more preferably at least 70%, are CD56 +.

The invention relates to the use of a population of CD56 ⁺ cells obtained according to the methods described above in the preparation of a cellular therapy product for the treatment in humans of post-ischemic heart failure or for repair of heart tissue. Indeed, the implementation of the method makes it possible to obtain a large number of cells rapidly and the cell populations obtained have the advantage of being homogeneous, and therefore particularly suitable for the preparation of a cell therapy product.

It also relates to the use of a population of cells from the same muscle biopsy and comprising from 500x10 ⁶ to 800 x 10 ⁹ cells of which at least 50%, and better still at least 60%, are CD56 + or HLA Class 1 in the preparation of a cellular therapy product for treatment in humans, post-ischemic heart failure or any genetic, viral, medicinal, infectious or parasitic heart disease

The treatment of heart disease consists in particular of injecting with a needle a population of cells, the dominant type has the characteristics of myoblastic cells, obtained and prepared as a cell therapy product, directly in the myocardial tissue (12) or indirectly in the arterial circulation (24).

Preferably, at least 600 x 10 ⁶ cells from the same biopsy are injected.

Heart failure is now managed by treatment with angiotensin converting enzyme (ACEI) inhibitors. The experiments described below indicate a definite improvement effect of myoblastic cell transplantation in situ in the infarcted zone when this transplantation is performed concomitantly with CIRA treatment.

The invention therefore also relates to the use of cell populations of muscle origin obtained according to the methods described above as a transplant for potentiating the pharmacological treatments of heart failure.

The experimental part of the present text describes the carrying out of autologous muscle cell transplantations in rats demonstrating the feasibility of this technique. Indeed, the results show that the transplantation of these cells in the rat significantly improves the functional evaluation parameters thus demonstrating the feasibility of such transplants. They also show that the improvement in myocardial function is related to the number of grafted cells.

The experimental part also describes the results obtained in the rat, during the joint use of autologous muscle cells and the pharmacological treatment of heart failure.

During the cell growth and expansion phases in the methods provided by the invention, a step of genetic modification of the cells by transfection of a heterologous nucleic acid can be performed. The nucleic acid is selected to allow expression of a polypeptide or protein in the transfected cells. The transfected cells are then transplanted and allow delivery of the polypeptide or protein expressed from the heterologous nucleic acid, wherein said polypeptide or protein is a biologically active product. The invention thus relates to the use of a cell population as a cell therapy product as a delivery platform for a biologically active product.

The first part presents the examples of implementation of the method according to the selected culture stage for obtaining cell populations whose dominant cell type is CD34 + or CD15 + or CD56 + (myoblastic) or double negative CD56- / CD15-.

The results presented in the second part show the efficiency of the technique of transplantation of myoblastic cells on tissues cardiac infarction in the rat. It also makes it possible to determine the criteria required for a good efficiency.

Finally, a third part presents human clinical trials of muscle cell transplantation for myocardial tissue reconstitution, myocardial tissue repair, metabolically active tissue generation, tissue generation with functional activity. does not exist before reconstitution. It also shows that muscle cells can also play a role in remodeling heart tissue in humans.

EXPERIMENTAL PART Legend of figures:

Figure 1 :

Graph showing the LVEF values between the processed group and the control group.

Figure 2 :

Graph showing the LVEDV values between the processed group and the control group.

Figures 3A and 3B :

Graph showing LVEF values at 1 month (3A) and LVEF at 2 months (3B) according to the risk categories.

Figure 4 :

Linear regression showing the correlation between functional improvement and the number of cells injected.

Figure 5 :

Pre-operative (A) and post-operative (B) echocardiograms: The systolic thickening of the posterior ankemic wall is clearly visible.

Figure 6 :

Horizontal view of the two ventricles by positron emission tomography imaging showing the posterior wall of the left ventricle (grafted area at the bottom of the scan). Photos 6A and 6B (top) show a homogeneous metabolic activity in the septal and anterior walls with a decrease in metabolic activity in the posterior wall before the operation. Photos 6C and CD (bottom) show the uptake of deoxyglucose ₂ fluoro ¹⁸ in the posterior wall after surgery.

Figure 7 :

Stability of cell population characteristics (CD56 +) as a function of successive expansions

A. METHOD FOR OBTAINING CELLULAR POPULATIONS FROM SKELETAL MUSCLE TISSUES A.1 Materials, solutions and media used

Medium A: Modified MCDB120 medium (23): substitution of L-Valine with D-Valine, removal of phenol red, removal of thymidine.

Medium B: Medium A + 20% irradiated fetal calf serum + antibiotics (at 100 IU / ml for penicillin and 100 μg / ml for streptomycin).

Medium C: Medium B + bFGF (10 ng / ml) + dexamethasone (1 μM).

Solution D: PBS

Medium E: Medium A + bFGF (10 ng / ml) + dexamethasone (1 μM) + sterile human serum albumin (0.5%).

Solution F: Sterile isotonic saline solution 0.9% NaCl.

Solution G: Solution F + 4% human serum albumin and 7.5% final DMSO (dimethyl sulfoxide).

Solution H : Injection solution F + 0.5% human serum albumin

• ■ prélèvement et éminçage de la biopsie
• ■ dissociation enzymatique et séparation des cellules indivualisées par filtration
• ■ mise en culture des cellules et phases d'expansion des cultures
• ■ identification des types cellulaires présents aux différents stades de la culture par l'analyse de marqueurs cellulaires spécifiques
• ■ récolte des cellules
• ■ préparation et/ou maintien en survie et/ou congélation des cellules
• ■ préparation du produit de thérapie cellulaire

The method for obtaining cell populations described below comprises 6 steps:

• ■ sampling and cutting of the biopsy
• ■ enzymatic dissociation and separation of indivualized cells by filtration
• ■ cell culture and crop expansion phases
• ■ identification of cell types present at different stages of the culture by the analysis of specific cell markers
• ■ harvesting cells
• ■ preparation and / or maintenance of the cells' survival and / or freezing
• ■ preparation of the cell therapy product

In the process, the cells are centrifuged at 160 g for 5 minutes. Cell counting and population analysis are performed using a Neubauer hemacytometer. In the expansion phases, the cells are incubated at 37 ° C. in an air-CO ₂ incubator (95% -5%) saturated with moisture. The cells are observed using an inverted phase-contrast microscope.

A.2 Results A.2.1 Biopsy sampling and slicing

The sample is taken in the operating room, in a sterile environment, in an open system. A biopsy of approximately 10-16 grams of skeletal muscle tissue is performed by the surgical team. The biopsy is then cut into small cubes of 2 to 4 mm on one side, then minced with fine scissors in medium A. The emine is placed in a sterile bottle containing 25 ml of medium A.

The slicing stage is also assisted, using Medimachine ® knife mills (distributed by Becton-Dickinson). In this device, fragments of mass less than 0.2 g are dissociated in the sterile Medicon receptacles, after grinding controlled by an electric motor, lasting less than 5 minutes. Repetition of the operation using several receptacles Medicon allows to finally prepare several grams of muscle. Table A below shows the proportions of the CD56 +, CD15 + and CD34 + cell types obtained during the different culture stages J0, J15, J20 and J26 (the proportion of CD34 cells being almost zero at J15, this one does not is not indicated in the table for the steps following the cultivation J0). It is observed that, unexpectedly, the biopsy cutting step is a crucial step in rapidly obtaining a population comprising a dominant CD56 + myoblast type cell type. TABLE A: SCISSING COMPILATION WITH SCISSORS / MECHANICAL BROYAT BIOPSY 1 BIOPSY 2 SCISSING WITH SCISSORS MECHANICAL BROYAT SCISSING WITH SCISSORS MECHANICAL BROYAT D0 WEIGHT 0.8 g 0.8 g 1.2 g 1.4 g CNT 10 * 5 2.6 2.4 6.4 4.8 % CD34 + 7.4 4.7 2.6 1 % CD56 + 0.4 0 1.2 0.4 % CD15 + 0 0 0.9 3.3 PASSAGE 1 DAY J15 J15 J7 J15 CNT 10 * 5 11.7 18.4 16.3 16.3 % CD56 + 51.6 90.5 35.1 77.9 % CD15 + 1.8 0.9 44.1 2.6 PASSAGE 2 DAY J20 J20 J21 J21 % CD56 + 58 93.4 15 89.6 % CD15 + 20.9 4.4 20 7.3 PASSAGE 3 DAY J26 J26 J27 J27 % CD56 + 43.6 75.8 49.7 60.2 % CD15 + 30.1 1.7 10.9 1.1

Tableau C : Cultures à partir de biopsies de poids inférieur à 0,5 g PASSAGE 1 PASSAGE 2 PASSAGE 3 PASSAGE 1 PASSAGE 2 PASSAGE 3 BIOPSIE J TRAITEMENT J0 J10 J14 J16 BIOPSIE J TRAITEMENT J0 J6 J11 J17 5007 CNT 10*5 1,2 11,2 64 266 5054 CNT 10*5 2,8 6,6 7,1 370 % CD34+ 28,8 ND ND 0 % CD34+ 10,7 ND ND ND 0,23 g %CD56+ 1,95 68,6 87,6 89,5 0,45 g %CD56+ 25,7 62,3 69,1 84,6 %CD15+ ND 30,9 17,3 28,8 %CD15+ ND 31 26,9 14,5 % VIABILITE 88,9 96 100 98,7 % VIABILITE 88,7 100 96 87 BIOPSIE J TRAITEMENT J0 J7 J10 J15 BIOPSIE J TRAITEMENT J0 J7 J8 J15 5008 CNT 10*5 2,04 7 21,6 586,6 5058 CNT 10*5 7,7 9,4 13,6 534 % CD34+ 34 ND ND 0 % CD34+ 44,3 21,4 ND ND 0,23 g %CD56+ 6,1 47,1 66,3 92,9 0,19 g %CD56+ 8,4 23,2 27,5 72,2 %CD15+ ND 44,6 30,1 9,5 %CD15+ ND 63,2 63,6 18,9 % VIABILITE 85,1 92 88 97,9 % VIABILITE 95 82,1 100 97 BIOPSIE J TRAITEMENT J0 J7 J10 J14 BIOPSIE J TRAITEMENT J0 J6 J13 J15 5011 CNT 10*5 2,1 9,8 35 285 5060 CNT 10*5 5,2 9 18,8 663 % CD34+ 26,2 ND ND ND % CD34+ 48,7 ND ND ND 0,2 g %CD56+ 3,5 24,1 29,9 38 0,33 g %CD56+ 7,9 42,3 52,8 89,7 %CD15+ ND 70,8 63,8 60,5 %CD15+ ND 46,8 48,6 11,9 % VIABILITE 85,5 98 98 99 % VIABILITE 94,4 100 99 98 Different sample sizes were tested for carrying out the process of the invention, covering a weight range of 0.13 g to 14.9 g. The results presented in the following tables show that the evolution of the proportions of the different types of population and the amplification of the number of cells evolve in a comparable manner for biopsy sizes of less than 1 g (Table B) to more than 10 g (Table C).

<b> Table C: Crops from biopsies weighing less than 0.5 g </ b> PASSAGE 1 PASSAGE 2 PASSAGE 3 PASSAGE 1 PASSAGE 2 PASSAGE 3 BIOPSY J TREATMENT D0 J10 J14 J16 BIOPSY J TREATMENT D0 J6 J11 J17 5007 CNT 10 * 5 1.2 11.2 64 266 5054 CNT 10 * 5 2.8 6.6 7.1 370 % CD34 + 28.8 ND ND 0 % CD34 + 10.7 ND ND ND 0.23 g % CD56 + 1.95 68.6 87.6 89.5 0.45 g % CD56 + 25.7 62.3 69.1 84.6 % CD15 + ND 30.9 17.3 28.8 % CD15 + ND 31 26.9 14.5 % VIABILITY 88.9 96 100 98.7 % VIABILITY 88.7 100 96 87 BIOPSY J TREATMENT D0 J7 J10 J15 BIOPSY J TREATMENT D0 J7 J8 J15 5008 CNT 10 * 5 2.04 7 21.6 586.6 5058 CNT 10 * 5 7.7 9.4 13.6 534 % CD34 + 34 ND ND 0 % CD34 + 44.3 21.4 ND ND 0.23 g % CD56 + 6.1 47.1 66.3 92.9 0.19 g % CD56 + 8.4 23.2 27.5 72.2 % CD15 + ND 44.6 30.1 9.5 % CD15 + ND 63.2 63.6 18.9 % VIABILITY 85.1 92 88 97.9 % VIABILITY 95 82.1 100 97 BIOPSY J TREATMENT D0 J7 J10 J14 BIOPSY J TREATMENT D0 J6 J13 J15 5011 CNT 10 * 5 2.1 9.8 35 285 5060 CNT 10 * 5 5.2 9 18.8 663 % CD34 + 26.2 ND ND ND % CD34 + 48.7 ND ND ND 0.2 g % CD56 + 3.5 24.1 29.9 38 0.33 g % CD56 + 7.9 42.3 52.8 89.7 % CD15 + ND 70.8 63.8 60.5 % CD15 + ND 46.8 48.6 11.9 % VIABILITY 85.5 98 98 99 % VIABILITY 94.4 100 99 98

Biopsies were taken from patients aged 15 to 73 years. The results presented in Table D below show that the method is applicable regardless of the age of the patient on which the biopsy is taken. Table D: Preparation of muscle-derived cells from biopsies from patients of different ages Day 0 First passage Second passage Third passage Last name Weight (g) Patient age (years) Number 10 * 5 % CD56 + Number 10 * 6 % CD56 + Number 10 * 6 % CD56 + Number 10 * 6 % CD56 + MYO1 14.9 73 100 3.2 19.5 48 315 58.3 890 67.3 MYO3 10.4 63 43 3.4 14.2 67.7 156 87.1 922 91.3 MYO4 13.9 67 102 32.3 3.4 76.9 115 97.5 657 97.1 MYO5 11.6 39 117 22.1 31 71.5 244 89.9 993 95.2 MYO6 12 55 164 28.7 26.5 82 483 91.3 1210 84.9 4929 0.19 15 1 ND 1.6 64.3 4.7 75.4 56 82.4 5008 0.24 45 2 6.1 0.7 47.1 2.2 66.3 59 92.9 MOS 3.6 84 110 ND 75 93.4 240 96.4 565 93.7 CEL 3.0 51 45 ND 42 51.8 120 63.7 548 68.7

Biopsies can be stored for 90 hours at 4 ° C or frozen in balanced saline before being cultured. The following Tables E and F show that the viability of the cells and the evolution of the proportions of the different types of population born are not significantly affected after 90 hours of preservation of the biopsy at 4 ° C., or after freezing. Table E: Placement of a preserved biopsy 90 h at + 4 ° C in a balanced saline solution and preparation of muscle cells according to the method WEIGHT: 1.05 g D0 PASSAGE 1 (J7) CNT 10 * 6 0.8 4.9 % CD34 + 7.7 NS % CD56 + 6.5 58.7 % CD15 + 8.4 37.7 % VIABILITY 90.5 95 NS: not significant PASSAGE 1 PASSAGE 2 PASSAGE 3 BIOPSY J TREATMENT D0 J18 J20 J25 4929 CNT 10 * 5 0.97 16.2 47.3 55? 7 thawed % CD34 + 20.5 ND 0.04 0 muscle not % CD56 + 44.9 64.3 75.4 82.4 pathological % CD15 + ND 27.1 24.5 24.5 WEIGHT: 0.19 g % VIABILITY 90.8 100 96.8 96.7

The culture method can be implemented from biopsies of healthy subjects or patients with pathology. The following results presented in Table G show in particular that the the invention can be implemented for the preparation of cells of muscular origin derived from a patient suffering from Duchenne muscular dystrophy. Table G: Culture from a thawed biopsy from a patient with Duchenne muscular dystrophy PASSAGE 1 PASSAGE 2 PASSAGE 3 PASSAGE 4 PASSAGE 5 BIOPSY STADIUM D0 J7 J14 J21 J26 J29 4964 CNT 10 * 5 1.58 1.78 11.6 473.6 2411.2 6397 DE 24 H % CD34 + 53.9 ND 0.3 0 ND 0 striated muscle % CD56 + 5 24.6 78.1 73.7 60 52.5 paravertébral % CD15 + 0.4 30.4 12.7 15.5 26.4 36.1 WEIGHT: 0.136 g % VIABILITY ND 94 82.5 91.2 95.6 98

The results presented below show that the method can be implemented from any type of muscle biopsies. In particular, biopsies were obtained from different muscles, the paravertebral, the anterior tibialis, the peroneal longus, the common extensor toes, the peroneal longus, the posterior tibialis and the soleus. The results obtained according to different biopsies are presented in Table H.

A.2.2 Enzymatic dissociation and separation of individualized cells by filtration

The vial containing the emine is centrifuged at room temperature. The supernatant is removed by aspiration. The weight of the emerald is obtained by weighing on a weighed scale using an empty bottle. The emulsion is rinsed with 25 ml of medium A. After sedimentation of the emulsions, the supernatant is removed by suction.

A liberase solution (Roche-Boehringer) was prepared according to the manufacturer's instructions and then reconditioned and frozen at a concentration of 10 mg / ml. The liberase is defrosted extemporaneously and then added to the emulsion at a concentration of 0.1 mg / ml, in a volume of 10 ml per gram of tissue. The bottle is placed in an oven at 37 ° C for a period of 60 minutes. The bottle is agitated manually every 5 to 10 minutes (diffusion of the enzyme and gentle mechanical dissociation). The suspension is then centrifuged. The supernatant is removed by pipetting. This first digestion product is then incubated in a 0.25% trypsin solution. 10 ml of enzymatic solution per gram of initial tissue is used. The suspension is digested for 20 minutes at 37 ° C. in an oven, with manual agitation every 5 minutes. It is then aspirated and discharged through a 25 ml pipette. 10% irradiated fetal calf serum (Hyclone) is then added for the neutralization of enzymatic activities.

The digestion product is filtered through a 100 μm sieve and then 40 μm under gravity pressure in order to separate the dissociated cells from the residual tissues. One sieve per gram of tissue is used (Falcon Cell strainer). The filtrate is centrifuged at 300 g for 5 minutes. After removal of the supernatant, the pellets are washed with medium B and then centrifuged. The supernatant is removed by aspiration. The pellet is then taken up in 10 ml of medium C. 100 μl of volume is taken for counting. An aliquot is reserved for estimating viability by cytofluorimetry (propidium iodide).

A.2.3 Cell culture and crop expansion phases

After deconditioning, the cells are transferred to the culture device. The culture device is a culture tray (Nunc Single Tray) with an area of 600 cm ² . The filling of the tray is done through a hole provided for this purpose and closed by a sterile single-use stopper. The cultures are incubated at 37 ° C. in a chamber saturated with humidity in an air-CO ₂ controlled atmosphere (95% -5%).

The day after the planting, a first emptying is carried out in order to eliminate the dead cells and the muscular debris. An empty bag is connected to one of the two orifices of the culture device. The medium is removed by gravity and replaced with 120 ml of medium C which are added to the culture. Medium C is renewed after 120 to 192 hours. The expansion is decided when the degree of confluence of the cells reaches 20% to 50% or when the first myotubes appear (approximately 8 days after the culturing).

After emptying the medium, the cells are washed by gentle manual stirring with 50 ml of solution D. The solution D is drained and then 20 ml of irradiated trypsin solution (0.25%) are added. The flask is incubated for 5 minutes at 37 ° C. The cells are harvested in a 40 ml bag. The action of trypsin is neutralized by the addition of 10% fetal calf serum. The serum is injected inside the bag using a syringe. The cells are centrifuged. The supernatant is removed by transfer to another emptying bag, connected by a sterile connector. The pellet is resuspended in 30 ml of medium C for washing the cells and then the cells are centrifuged. The supernatant is removed by transfer to another emptying bag, connected by a sterile connector. The cell pellet is resuspended in 20 ml of medium C. An aliquot is taken for counting and analysis of the populations. Viability is estimated using a cytofluorimeter. The cells are then transferred to a bag containing 500 or 750 ml of medium C, then seeded in two or three double-tray units (Nunc double-tray) with a total surface of 1200 or 1800 cm ² . The cells are incubated. A third expansion is decided when the degree of confluence of the cells reaches 60% to 70% or when the first myotubes appear. For this series of expansions, multi-stage boxes of 10 trays (Nunc multi-tray) are used. After emptying the medium, the cells are washed with 100 ml of solution D. The detachment of the cell layer and the enzymatic dissociation of the cells are carried out after draining the washing solution by the addition of 50 ml of trypsin irradiated (0.25%) in each tray. The preparations are incubated for 5 minutes at 37 ° C. The cells are harvested in sterile pockets of 300 to 600 ml. The action of trypsin is neutralized by the addition of 10% by volume of fetal calf serum. The cells are centrifuged and the supernatant is removed by connection to a drain bag.

The cell pellet is resuspended in 50 ml of medium C and then transferred into one or two vials containing 1200 or 2400 ml of medium C by a sterile connector. One or two culture dishes of 10 multi-stage trays (multi-tray Nunc) is seeded with 1200 to 2400 ml of the cell preparation. Visual control of growth in multi-stage trays is impossible. Therefore, a single sterile Nunc culture dish (single-tray) is also seeded with 110 ml of the cell preparation. This last plate allows a daily visual control of the culture and the state of confluence of the cells. Cells are then incubated. The culture lasts 3 to 5 days. The day before harvesting the cells, the medium is removed by emptying into a sterile bag and replaced by an equivalent volume of medium E. The final harvest is decided when the degree of confluence of the cells reaches 90% or when the first myotubes appear.

A.2.4 identification of cell types present at different stages of culture by analysis of specific cell markers

Characterization is based on flow cytofluorometric analysis (FACS). Antibodies to the following human cell surface antigens were used: CD5, CD10, CD11, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD28, CD31, CD34, CD38, CD40, CD40-ligand, CD44 , CD45, CD56, CD62, CD71, CD80, CD86, CD90, CD105, CD117, CD138. Antibodies directed against the following antigenic structures have also been used: CD138, HLA-Class I, HLA-DR, ELAM, ICAM, LECAM, Stro-1, S-endo-1, VCAM, VLA2, VLA3, VLA4, VLA5, VLA6.

• Après mise en suspension dans du PBS, les cellules sont fixées et perméabilisées par l'addition de 10 volumes de méthanol à 4°C pendant 5 minutes, puis centrifugées. Après lavage du méthanol résiduel et centrifugation, les cellules sont reprises dans du PBS contenant l'anticorps dirigé contre la desmine (Dako, clone D33, 1/100) et incubées durant 15 minutes. Après lavage et centrifugation, les cellules sont incubées durant 15 minutes en présence de l'anticorps secondaire dirigé contre l'anticorps primaire, couplé à un fluorophore. Après lavage et centrifugation, les cellules sont analysées par FACS. Le tableau I ci-après présente les caractéristiques des marqueurs principaux utilisés et les types cellulaires correspondants (types cellulaires).

The analysis of the expression of desmin, intracellular protein is carried out as follows:

• After suspending in PBS, the cells are fixed and permeabilized by the addition of 10 volumes of methanol at 4 ° C for 5 minutes and then centrifuged. After washing the residual methanol and centrifugation, the cells are taken up in PBS containing the antibody directed against desmin (Dako, clone D33, 1/100) and incubated for 15 minutes. After washing and centrifugation, the cells are incubated for 15 minutes in the presence of the secondary antibody directed against the primary antibody, coupled to a fluorophore. After washing and centrifugation, the cells are analyzed by FACS. Table I below presents the characteristics of the main markers used and the corresponding cell types (cell types).

CLASSICAL CHARACTERISTICS OF MARKERS USED

Table I : Presentation of certain cellular markers used in the process and the cell types expressing them. MARKER PEN CELLULAR TYPES CD10 preB lymphocytes. neutrophil CD13 monocytes, myeloid cells CD15 monocytes, macrophages, granulocytes, eosinophils CD16 NK, under pop. T lymphocytes, neutrophils CD34 progenitors CD38 LT activities, stem cell, under pop. L T, B, NK CD40 activated CD4 + T cells CD44 Anti HCAM CD45 leukocytes CD56 NK, under pop. T cells CD71 proliferating cells CD117 progenitors HLA CL1 MHC class 1 antigen HLA CL2 MHC class 2 antigen Vla3 B lymphocytes VLA5 memory T cells, monocytes, platelets VLA6 thymocytes, memory T cells, monocytes desmin muscle cells

• Au jour J0 correspondant à la mise en culture de la population fraîchement préparée.

The analysis of the cell populations is carried out at the different stages of culture:

• Day D0 corresponding to the cultivation of the freshly prepared population.

• Au jour 1 (J1), la fraction non adhérente (surnageant) et la fraction adhérente (obtenue par trypsination) ont été analysées indépendamment. De manière intéressante, il a été montré que ces deux fractions renferment des populations aux caractéristiques différentes.
• De J2 à J9, les cellules adhérentes ont été analysées chaque jour afin de suivre l'évolution des types cellulaires au cours du temps.

Several series were carried out in unit bottles cultured parallel to the single plate unit at D0, in order to be able to monitor the evolution of the cultures daily by taking one or more bottles per day.

• On day 1 (D1), the non-adherent fraction (supernatant) and the adherent fraction (obtained by trypsination) were analyzed independently. Interestingly, these two fractions have been shown to contain populations with different characteristics.
• From day 2 to day 9, the adherent cells were analyzed daily to follow the evolution of cell types over time.

In parallel, the cells obtained by mass production were analyzed at each expansion stage and then at the time of the final harvest. The results showed that at equivalent harvest date, the characteristics obtained in the trays and the independent flasks are identical.

Data from the identification of cell types during differentiation kinetics are presented in the following Tables JA and JB and the essential characteristics are described below.

Kinetics of differentiation (percentages)

Table JA : Identification of cell types over time (as a percentage in the total cell population) of stages J0 to J3 of the culture. MARKERS D0 J1 SN J1 J2 J3 CD34 + 38.5 (25-75) 77 (69-79) 39 (25-67) 74 (64-75) 74 (51-75) CD34- 61.5 (25-75) 23 (20-30) 61 (33-75) 26 (25-30) 26 (25-49) CD34 + CD10 + 14 (10-18) 27 (25-29) 2.5 (2-3) 24 (20-40) 11 (7-17) CD34 + CD10- 25 (23-31) 47 (40-54) 30.5 (24-37) 39 (36-54) 59 (44-63) CD34 + CD45 + <5 ND 0.5 (0-1) ND ND CD34 + CD56 + 0 0 0 0.3 (0.3-0.3) 0.7 (0.4-2) CD34 + DR + 14 ND 21.5 (16-27) ND ND CD34 + DR- 28 ND 12 (12-12) ND ND CD34 + CD 15+ ND ND ND ND ND CD56 + 4.7 (0-26) 4.4 (0.3-15) 1 (0-5) 11 (7.7-20) 14 (12-35) CD15 + 1.5 (0-6) 3 (1,5-6) 3.6 (0.1-12) 20.5 (20-21) 25 (24-33) CD56 + CD15 + 0.02 (0-0.3) 0 0 4 (4-4) 10 (6-14) CD13 + ND ND ND ND ND CD44 + 0 ND ND ND ND CD117 + 1.7 (0.16-1.7) ND 1.7 (0.2-2) ND ND Table JB : Identification of cell types over time (as a percentage in the total cell population) of the J4 to D8 stages of the culture. MARKERS J4 J5 J6 J7 J8 CD34 + 56 (36-67) 35 (26-50) 23 (13-47) 2.7 (0.6-10) 1.7 (0.4-3.4) CD34- 44 (33-63) 65 (50-73) 76 (53-87) 97 (90-99) 98 (96-100) CD34 + CD10 + 9 (8-22) 16.5 (3-34) 16.8 (0.6-33) 1.7 (0.2-2.7) 0.4 (0.2-7.6) CD34 + CD10- 35 (27-59) 25 (15-35) 14 (14-14) 5.1 (0.4-9) 1.3 (0.2-7.6) CD34 + CD45 + ND ND ND ND ND CD34 + CD56 + 3 (1,5-6) 7.5 (1.5-12) 3 (0.1-12) 0.35 (0.2-0.6) 0.4 (0.4-2.1) CD34 + DR + ND ND ND ND ND CD34 + DR- ND ND ND ND ND CD34 + CD15 + ND ND ND ND ND CD56 + 34 (28-55) 61 (50-68) 73 (57-74) 64 (52-87) 68.4 (50.3-91) CD15 + 48 (46-48) 48 (47-63) 51 (29-64) 36 (20-55) 29 (12-54) CD56 + CD15 + 19 (17-21) 13 (9-25) 10 (5-13) 3.6 (2.6-6) 1.4 (1-3) CD13 + ND ND 96 (76-97) 99 (94-99) 99 (96-99) CD44 + ND ND ND ND ND CD117 + ND ND ND ND ND

A.2.4. 1 Characteristics of cell preparations at day 0

The method makes it possible to obtain at ³ × ^{10 5} to 4 × 10 ⁶ cells per gram of tissue. The majority cell types are CD34 +. The CD34 + cell types are CD34 + / CD10 + at 14%; CD34 + / CD10- at 25%; CD34 + / CD56 + at 0%; CD34 + / DR + at 14% and CD34 + / DR at 28%. The CD34 + populations are predominantly CD45-. Minority populations express CD44, CD45, CD56, CD117, HLA-Class II. Surprisingly, the characterization of a large number of progenitor cells (in absolute and relative amounts in the population) makes it possible to envisage, by harvesting the cells at this stage, their use as a cell therapy product for the reconstitution of numerous cells. tissues, not only muscle but also hematopoietic, bone, adipose, cartilaginous or vascular.

A.2.4.2 Characteristics of adherent cell preparations at J1

A majority of cells is CD34 +. The population consists of CD34 + / CD10 + (27%) and CD34 + / CD10- (47%). CD13 appears. The preparation is negative for CD117 and CD45. The CD15 + and CD56 + cell types are in the minority.

A.2.4.3 Characteristics of cell preparations present in the supernatant on D1.

A significant proportion of cells is CD34 +. The population consists mainly of CD34 + / CD10-. A CD117 + population (<5%) is present and expresses CD45 +. Some minority populations are present: CD38 + (15%), CD45 +, few CD15 + and CD56 +, HLA-Class II +.

A.2.4.4 Characteristics of changes in markers during culture

The evolution is marked by an increasing increase in the proportion of CD15 + and / or CD56 + cells and a decrease in CD34 + populations. The gradual transition from a CD34 + to CD15 + population can be observed over time. The proportion of CD13 +, CD44 +, CD71 + populations increases with time. There are minority populations CD138 +, HLA-Class II +, CD38 +.

At the end of the expansion, there are three predominant populations: CD56 +, CD15 + and CD56- / CD15-. The CD56 + population expresses CD10, CD13, CD44, desmin and HLA-Class I. These markers are specific for myoblastic cells. The CD15 + population expresses CD13, Class I and partially CD10. In the CD56- / CD15- population, one fraction expresses desmin and the other fraction does not express it. Some markers are expressed more weakly and variably: CD71, VLA3, VLA5, VLA6, CD16 + and CD40L. The CD34 +, CD38 +, CD45 + and HLA-Class II + populations have disappeared or are extremely minor.

A.2.4.5 Cell characteristics after depletion of the CD34 + fraction

Table K below represents the characteristics of the cells obtained at the end of the first pass, by comparing different initial conditions. Eight independent experiments are represented. After depletion of the CD34 + fraction, the method makes it possible to rapidly obtain a cell population that is predominantly composed of cells expressing CD56. In particular, the proportion of cells expressing CD56 is greater than that obtained from an unexplained biopsy. <u> Table K. Depletions or enrichments in CD34 + cells present in muscle biopsy. </ u> % CD56 + at first pass Experience 1 Experience 2 Experience 3 Experience 4 Experience 5 Experience 6 Experience 7 Experience 8 Non Separated Fraction ND 72% 87% 41% 48% 67% 70% 38% Fraction Deployed in CD34 93% 98% 97% 82% 93% 93% 86% 57% Fraction Enriched with CD34 61% 36% 37% 1% 8% 28% 4% 12%

A.2.5 Harvesting cells

The following protocol describes harvesting the cells at the final stage to obtain a majority myoblastic population. However, the protocol allows the person skilled in the art to implement it whatever the stage of differentiation chosen for harvesting the cells and this, depending on the desired cell population.

After emptying the medium, the cells are washed with 500 ml of solution D (for the multi-tray units), 50 ml for the single unit and 100 ml for the double units. The wash solution is drained and 200 ml of irradiated trypsin solution (0.25%) is added to the multi-tray units (20 ml for the single unit, 40 ml for the double units). The preparation is incubated for 5 minutes at 37 ° C. The cells are collected by emptying in a 500 ml bag. The action of trypsin is neutralized by the addition of 10% fetal calf serum injected by a syringe. The cells are washed as follows: the cells are centrifuged. The supernatant is removed and the cells are resuspended in 300 ml of solution F and centrifuged. Supernatants are eliminated. Two other washing steps as previously described are carried out. These successive washes are intended to remove trypsin, animal proteins still present and recombinant bFGF. During the third wash, an aliquot is reserved for cell count, viability and cell quality estimation, microbiological quality controls. The cells can be concentrated in solution H so as to obtain a suspension that is adequate for the clinical use requested.

After centrifugation, the cells are taken up in a volume of isotonic solution at a concentration of 1.5 × 10 ⁸ cells / ml. They are finally sucked into a 10 ml syringe. The cells are removed for injection into sterile syringes. The type of needle used for the injection depends on the targeted tissue. For direct intramyocardial injection, a 90 ° 25 to 30 gauge bent needle is specifically used.

A.2.6 Production yields and characteristics of cell types

Table L below summarizes the results obtained during the implementation of the method of the invention from different biopsies obtained from three different patients.

The cells were initially produced in single, double and multiple plate units until the third included expansion. The expansions were subsequently carried out by duplicating the populations and transplanting them into culture dishes of 25 cm ² . At each pass, the majority of the cells were used for characterization and counting, while a known number of cells were used for duplicate seeding. The number of cumulative doublings allows, by calculation, to obtain about 100 billion cells between the eighth and the ninth expansion.

L'histogramme à la figure 7 présente les valeurs médianes d'expression de CD56 et de CD15 sur 8 échantillons au cours des différentes phases d'expansions.Table L shows the yields in terms of number of cells obtained and in terms of proportion of CD56 + or desmin + cells in the population at the different expansion phases for the three patients named MYO 003, MYO 004 and MYO 005.

The histogram at the figure 7 presents the median expression values of CD56 and CD15 on 8 samples during the different phases of expansions.

The results presented at figure 7 show that the proportions of the CD56 + and CD15 + cell types obtained are relatively homogeneous according to the different biopsies, and are not modified during the different expansions. In particular, it is found that the CD56 + type cells remain in a dominant proportion during the expansion phases.

These results further show that after identification of optimal harvest stages according to the desired cell types, a person skilled in the art can reproduce the process of the invention by dispensing with the cell characterization step as defined in FIG. method of the invention and by multiplying the expansion phases so as to obtain a large number of cells comprising a specific cell type in a dominant proportion, and in particular the CD56 + cell type.

A.2.7 Freezing the cell therapy product

In order to allow the cells thus prepared to be used over time, it may be advantageous to freeze them under conditions such that subsequent thawing allows sufficient cell survival, preferably greater than 90%.

By way of example, the cells are suspended in the freezing medium (solution G) and transferred to two sterile freezing bags at a concentration of between 10 ⁷ and 2 × 10 ⁷ cells / ml or in cryocongesting tubes at a concentration of between 1 x 10 ⁶ and 5 x 10 ⁶ / ml. The freezing is carried out using a device (Digicool or Nicool) ensuring a progressive descent in temperature controlled. The cells are stored in liquid nitrogen until defrosting.

Thawing of the cells is carried out in a water bath at 37 ° C. Cell preparations are washed twice with isotonic saline. The rinses are performed by sterile connection to the pockets of isotonic solution and the emptying pockets. An aliquot is reserved for estimating viability and cell quality.

B. FACTORS AFFECTING THE FUNCTIONALITY OF TRANSPLANTATION OF AUTOLOGOUS MUSCLE CELLS FOR THE TREATMENT OF MYOCARDIAL ISCHEMIC MODELS B.1 Materials and methods B.1.1 Model of myocardial ischemia

Wistar male rats weighing 280 g were anesthetized with ketamine (50 mg / kg) and xylasin (10 mg / kg) and ventilated by the trachea. A thoracotomy was performed. Myocardial infarction is obtained by left coronary ligation using 7/0 polypropylene wire.

B.1.2 Functionality tests

One week after myocardial infarction, and one or two months after transplantation, left ventricular function was studied by two-dimensional (2D) echocardiography.

Under general anesthesia with ketamine (50 mg / kg) and xylasin (10 mg / kg), the 2D (and M-mode) measurements are made with a 15MHz (15L8) linear-array transducer (Sequoia) , Acuson Corp., Mountain View, CA, USA) allowing a maximum frequency of 160 Hz. Longitudinal parasternal views are obtained, so that the mitral and aortic valves and the apex can be correctly visualized and thus recorded.

The length measurements (L) of the long axis of the left ventricle and the plots of the endocardial zones (a) are carried out. The end diastolic volume of the left ventricle (LVEDV) and the volume at the end of left ventricular systole (LVESV) were calculated with the following formula: V = 8x A ² / (3 x π x L). The left ventricular ejection fractions (LVEF) are thus calculated: LVEF = (LVEDV-LVESV) / LVEDV. All measurements were made from at least three beats and with the aid of two experimenters treating the different groups blindly.

B.1.3 Cell culture

During the myocardial infarction procedure, the anterior muscles of the right and left tibialis are dissected to separate the tendon and the aponeurotic tissue from the muscle tissue. They are then minced, weighed and dissociated with enzymes using collagenase IA (2 mg / ml, Sigma Chemical Co., St. Louis, MO, USA) for one hour and trypsin-EDTA (0.25%). , GIBCO BRL, Gaithersbueg, MD, USA) for 20 minutes.

The cells are harvested by sedimentation (7 min at 1200 rpm) and the enzymatic reaction is neutralized by the addition of 10% fetal calf serum. After passing through a 100 μm sieve and centrifugation, the supernatant is removed and the cells are resuspended in a medium composed of F12 (HAM) with 20% fetal calf serum, 1% (vol / vol) penicillin-streptomycin (10,000 IU / ml-10000 μg / ml, GIBCO BRL) and 5 μg / ml bFGF (Sigma).

The initial seeding is carried out in culture flasks of 75 cm ² and the cells are incubated in air saturated with moisture at 5% CO ₂ .

On day of transplantation, after 7 days of culture and after baseline functional evaluation of ventricular ejection fractions by echocardiography, the cells were harvested by trypsinization, washed and viability tested. A sample was seeded on 12-well dishes in 2.0 ml of culture medium for counting. The cells are washed in the injection medium (culture medium + 0.5% BSA, fraction V) and kept in the ice before transplantation. The cells are centrifuged, resuspended in 150 .mu.l of injection medium and administered sub-epicardially in the infarcted zone.

B.1.4 Transplantation of cells in the infarcted zone

Forty-four rats were included in this study and were divided into two groups: a control group and a treated group.

All rats were reoperated one week after myocardial infarction, under general anesthesia and tracheal ventilation. All rats received 150 μl of injection medium administered in the infarcted zone using a 30-gauge needle. In the control group (n = 23), the rats received the injection medium alone. The treated group (n = 21) received the suspension of cultured myogenic cells.

In each group, four risk categories were studied in relation to the LVEF baseline ejection fraction: <25% (n = 15), 25-35% (n = 15) and> 40% (n = 16) . This stratification makes it possible to obtain homogeneous numbers of animals in each subgroup in order to make the statistical results more precise.

B.1.5 Immuno- and histochemical studies

One day after transplantation, the cells seeded in a 12-well dish are fixed with methanol and cooled at -20 ° C. for 5 minutes. Non-specific staining is neutralized using a mixture of 5% horse serum (HS) and 5% fetal calf serum in PBS for 20 minutes. The cells are incubated with a mouse antibody directed against desmin (1/200 DAKO, A / S-Denmark) for 1 hour and then with an anti-mouse antibody conjugated to Cy3 marker (1/200, Jackson Immuno Research Laboratories, Inc. .) for an hour in the dark.

The cells are observed using an inverted phase contrast and fluorescent illumination microscope. Several snapshots were taken randomly. The proportion of myoblasts is calculated by dividing the number of desmin-positive cells by the total number of cells observed.

Within three months after the last echocardiogram (ie 2 months after transplantation), the rats are sacrificed by an overdose of ketamine and xylasin. The ventricles are isolated and divided into two parts along their longitudinal axis. Both parts are put in isopentane and frozen with nitrogen. Thin 8 μm sections are prepared using a cryostat and standard histological studies are performed by hematoxylin and eosin staining.

B.1.6 Statistical analysis

All data are on average ± 1 SEM. All assays were performed using the appropriate software (Statview 5.0, SAS Institute Inc., Cary, NC, USA). The critical threshold α for the analyzes was chosen at p <0.05.

Comparisons of continuous variables between the control and treatment groups and each risk category were performed using analysis of variance (ANOVA method) followed by the post hoc test (Scheffé). Longitudinal studies comparing echocardiographic data for each group before and one and two months after intramyacardial injection were performed using the matching test.

To test the relationships between the number of cells injected and cardiac function after transplantation, two variables were created: (LVEF at 1 month / LVEF) and (LVEF at 2 months / LVEF). The link was studied by calculating the F-ratio for the ANOVA regression and the adjusted R ² coefficient for the linear regression analyzes.

In addition, the variability within each group of echocardiographic tests was observed from two batches of measurements on 10 randomly selected rats using Bland and Altman analysis.

B.2 Results B.2.1 Characterization of the injected suspension

Of the 10,000 cells counted on the day of transplantation, 50% are positive for the expression of desmin. The number of injected cells is 3,500,000 ± 500,000 ranging from 700,000 to 6.5x10 ⁶ .

B.2.2 Functional test after transplantation of muscle cells

The baseline echocardiographic parameters are not significantly different between the groups. On the contrary, major differences between groups are observed after transplantation. Thus, in the treated group, cardiac function is improved, as shown by comparison of LVEF with the control groups ( Figure 1 ). At one month after myocardial injection, significant differences were observed between the two groups (37.52 ± 1.92% vs. 25.49 ± 2.47%, p = 0.0005). This result is confirmed after 2 months after the injection (40.92 ± 2.17% against 25.83 ± 2.39%, p <0.0001). This improvement in LVEF in the treated group is essentially related to a smaller increase in LVESV compared to ventricular dilatation, represented by the variable LVEDV, which increases similarly in both groups ( Figure 2 ).

Longitudinal analyzes in each group showed a substantial improvement in left ventricular function in the treated group ( Figure 3 ). Significant differences were observed comparing LVEF at one month and LVEF at 2 months at baseline LVEF variable. The differences are also significant when comparing LVEF at one month to LVEF at 2 months. While LVEDV and LVESV are increased to one month by comparing to baseline values (p <0.0001 and p = 0.0003, respectively) and at 2 months (p <0.0001 and p = 0.029, respectively), a stabilization and a decrease are shown when 2-month values are compared to 1-month values (p = 0.78 and p = 0.12, respectively). In the control group, a significant decrease in LVEF with a significant increase in LVEDV is already visible at one month (p = 0.0066 and p <0.0001 vs baseline respectively). Both parameters indicate values similar to 2 months.

When cardiac function (LVEF) is analyzed by hazard categories according to LVEF baseline values, differences are observed between control and treated groups at one month in the two intermediate 25-35% and 35-40% subgroups. This improvement in LVEF is confirmed at 2 months after injection in both subgroups, but interestingly, a beneficial effect is also observed, comparing the treated group to the control group in the risk group <25%.

Finally, the regression study shows a significant link between the number of grafted myoblasts and the LVEF ratios at one month (R ² = 0.675, p <0.0001) and at 2 months (R ² = 0.714, p <0.0001) ( Figure 4 ). When the data are analyzed by risk group, the impact of the number of cells injected at 2 months is also significant in the subgroups <25%, 25-35% and 35-40% (R ² = 0.836, p = 0.0106, R ² = 0.928, p = 0.0083 and R ² = 0.985, p = 0.0076, respectively).

B.2.3 Cumulative effects of muscle cell transplantation and treatment with an angiotensin converting enzyme (ACEI) inhibitor

The management of heart failure currently consists of the administration of ACE inhibitors. It was therefore interesting to study whether there could be a synergy between the transplantation of cells of muscle origin obtained according to the method of the invention and the protective effect provided by the ACE inhibitors.

Myocardial infarction was performed in 39 rats by ligation of the coronary arteries. Perindoprilat treatment at 1 mg / kg daily, an ACE inhibitor, was initiated immediately after infarction and continued without interruption until the animal was sacrificed. One week after the infarction, the animals were operated on again and randomly selected to receive a subepicardial injection of 150 μl of culture medium alone (control group, n = 21) or an equivalent volume containing the Muscle origin, approximately 3x10 ⁶ cells cultured from anterior tibia muscle biopsy and harvested at the time of infarction (treated group, n = 18). Left ventricular function was tested by echocardiography one month after transplantation. The baseline ejection fraction value is similar between control animals (24 ± 2%) and treated animals (28 ± 1%) (p = 0.11). One month after transplantation, the values of ejection fractions increased in both groups and were 32% ± 2% for control and 38% ± 2% for the treated group. However, there was a greater increase in the treated group (p <0.0001 vs. baseline) compared to the control group (p = 0.004 vs. baseline), with the ejection fraction value being significantly higher in the rats. treated (p = 0.04 against the control group). Analysis of the volume data showed that the functional improvement provided by muscle-derived cell transplantation is primarily related to an increase in contractility rather than a change in the left ventricle.

These data show that there is an additive effect of muscle cell transplantation and treatment with ACE inhibitors.

C. CLINICAL TRIALS IN MAN OF TRANSPLANTATION OF MUSCLE CELLS FOR THE RECONSTITUTION OF MYOCARDIAL TISSUES

Clinical trials of muscle-derived cell transplants prepared according to the method of the invention have been carried out in humans to treat heart failure.

C.1 Methods

The trials were conducted in 6 patients, of whom, at the time of inclusion, the age was between 18 and 75 years. These patients have all had clinical indications of coronary artery bypass grafting with the technical possibility of surgical revascularization. Their left ventricular ejection fraction (measured by echocardiography and / or angiography and / or isotopes) was less than or equal to 35%. They all had a history of transmural myocardial infarction. Finally, the patients had segmental hypo or akinesia affecting two or more contiguous segments (outside an aneurysm), related to the infarct. Residual metabolic activity in this area was less than 50%, detected by positron emission tomography (PET) and there was no increase in kinetics on low dose Dobutamine ultrasound (10 gamma / kg / min).

Ten to eighteen grams of autologous tissue are collected from the vastus lateralis in the patient. The incision is made in line with the external vastus. The protocol for culturing and expanding the cells is that described in Part A of this text.

The cell culture to be injected is then deposited in a sterile stainless steel cup to be sucked into a 1 ml syringe. A coronary bypass is first performed under extra-corporeal circulation according to the usual technique. After analysis of the extension of the infarct and identification of the edges of the necrotic zone, the cell suspension of approximately 650 to 1200 million cells (1.5x10 ⁸ cells / ml) is injected into and at the confines of the zone. infarction, using the 1 ml syringe. Several injections are necessary to deposit all the cells. This operating time is performed under extracorporeal circulation and clamping.

C.2 Evaluation methods and functional results

These tests have demonstrated the feasibility and safety of the technique in humans. In addition, functional tests are performed by measuring left ventricular function (segmental and global) and ventricular remodeling, coupled with the measurement of metabolic cellular activity. These measurements are obtained by ultrasonic methods such as the usual Doppler echocardiography (measurement of diameters, volumes and left ventricular ejection fractions), tissue Doppler in the infarcted zone and Dobutamine echocardiography for the investigation of ischemia and viability. They are also obtained by isotopic methods such as positron emission tomography (uptake of deoxyfluoroglucose in the infarcted zone).

These measurements are performed preoperatively and postoperatively at ¹ month ± 8 days and the 3 ^rd month ± 8 days.

The results obtained after transplantation of myoblasts on a patient are presented below.

The patient is a 72-year-old man hospitalized for heart failure (NYHA Class III) following an extensive inferior infarction of myocardial infarction that is refractory to medical treatment, especially since beta-blockers and angiotensin-converting enzyme inhibitors have had to be interrupted because not tolerated. The extracardiac balance also notes a moderate renal insufficiency (creatinine: 200 mmol / L) and a bilateral carotid occlusion without functional repercussion to the intracranial Doppler and therefore not an autonomous vascular gesture.

On echocardiography, the left ventricular ejection fraction is 20% with extensive akinesia of the lower wall and severe anterolateral hypokinesia. The lack of viability in this lower wall is demonstrated by the persistence of akinesia under low dose dobutamine. The anterolateral wall, on the other hand, has a biphasic response to dobutamine (low dose, then high dose) indicating viability and ischemia. These data are confirmed by positron emission tomography (PET) with ₂ fluoro ¹⁸ deoxyglucose (FDG), which shows that viability is totally lacking in the lower wall, but is present in the anterior and lateral portions of the ventricle. left.

1. (1) une altération importante de la fonction ventriculaire gauche,
2. (2) une cicatrice d'infarctus akinétique et métaboliquement non viable,
3. (3) une indication élective de pontage sur les artères autres que celles de l'infarctus.

On the coronarography, there is a complete, proximal occlusion of the anterior interventricular artery, with a late opacification of the downstream bed by ipsilateral collaterals, a tight stenosis of high diagonal branch and proximal occlusion of the right coronary artery. The right coronary artery has only insignificant irregularities. The present patient summarizes:

1. (1) significant impairment of left ventricular function,
2. (2) an akinetic and metabolically unsustainable myocardial infarction scar,
3. (3) an elective indication of bypass on the arteries other than those of the infarct.

Under local anesthesia, a fragment of the vastus externus is removed from the patient through a short incision (5 cm). The myoblasts are produced according to the method of the invention described in part A. The number of cells is multiplied using several expansions in multi-stage trays to obtain in two weeks 800x10 ⁶ cells, 65% of cells CD56 +. The proportion of viable cells is greater than 96%.

Two weeks after the biopsy, the patient is readmitted to the cardiac surgery department for bridging. Given the precariousness of the hemodynamic state after anesthetic induction (cardiac output at 1.5 Umin with a 55% oxygen venous saturation) an intraaortic balloon counterpulsation is instituted prophylactically. The anterior diagonal and interventricular arteries are bridged with a saphenous vein graft and the left internal mammary artery, respectively, under extracorporeal circulation and warm, retrograde, continuous blood cardioplegia. After completion of the coronary anastosomes, the infarcted area of the lower wall is easily identified. Thirty-three cell injections, suspended in 5 ml of albumin, are made in and around whitish foci of necrosis using a right-angle 27 G needle specially designed to allow the creation of subepicardial channels. almost phlyctenular in appearance. The duration of aortic cross-clamping is 56 min, of which 16 are dedicated to cell injections. There is no bleeding from injection sites, and extracorporeal circulation can be stopped without difficulty. The patient is weaned from the counterpulsation and pharmacological support with dobutamine in postoperative first 3 days and leaves the ^8th day after uneventful.

Eight months later, his clinical condition improved, and he is currently in NYHA Class II, while medical treatment has remained unchanged. A 24-hour Holter shows no disturbance of rhythm. Studies of 4 echocardiograms performed each month since the surgical operation show that the left ventricular ejection fraction has increased to 30%. As shown in figure 5 Segmental contractility is demonstrated not only in the anterior wall, but also in the contracting infarcted and grafted posterior zone (the percentage of systolic thickening has gone from preoperative values almost undetectable to 40%).

Again, this contractility is still improving under dobutamine. In addition, tissue Doppler imaging shows the appearance of a transmyocardial velocity gradient in systole. A new FDG positron emission tomography clearly shows the uptake of the tracer in the lower wall, with a ratio of activity between wall and septum (taken as a control), which increases from 0.5 before the operation to 0 , 7, which reflects a new metabolic activity in the infarcted zone devoid of preoperative viability ( Figure 6 ). This last observation can not have been influenced by the associated myocardial revascularization and, confronted with the echocardiographic data, suggests that the functional improvement in the infarcted zone is really related to the presence of grafted myoblasts.

Taken together, these results show that the function of the infarcted zone was improved by the transplantation of myoblasts obtained according to the method of the invention.

• Stade NYHA
• Fraction d'éjection
• Contractilité segmentaire

• III -> II
• 25 % -> 35%
• Augmentation (+/-)

Évolution viabilité segmentaire Amélioration (+) Nom du patient E. MYO 4 Age 67 Sexe M Type de Pathologie Insuffisance cardiaque post-ischémique Fraction d'éjection 31 % Stade classification NYHA III PET-Scan Présence d'une zone non viable Échographie initiale Akinésie apicale, akinésie postérieure, hypokinésie latérale Bilan virologique Négatif Durée de culture 20 J Cellules : nombre, caractéristiques 657 x 10⁶. CD56+ : 97 % ; CD15+ : 15 % ; Desmine+ : 58 % ; HLA Classel : 94 % ; Viabilité : 98 %; Formation de myotubes (test fonctionnel) : +. Siège d'injection Postérieure ; 620 x 10⁶ cellules injectées Complications infectieuses Absence complications infectieuses post-opératoires Évaluation fonctionnelle :

• Stade NYHA
• Fraction d'éjection
• Contractilité segmentaire

• III -> II
• 31 % -> 50%
• Augmentation (+/-)

Évolution viabilité segmentaire Amélioration (+) Nom du patient F. MYO 5 Age 39 Sexe M Type de Pathologie Insuffisance cardiaque post-ischémique Fraction d'éjection 22 % Stade classification NYHA III PET-Scan Présence d'une zone non viable Échographie initiale Akinésie apicale, akinésie postérieure, hypokinésie antérieure, hypokinésie latérale Bilan virologique Négatif Durée de culture 14 J Cellules : nombre, caractéristiques 993 x 10⁶. CD56+ : 95 % ; CD15+ : 4 % ; Desmine+ : 78 % ; HLA Classel : 96 % ; Viabilité : 98 %; Formation de myotubes (test fonctionnel) : +. Siège d'injection Postéro-latérale ; 950 x 10⁶ cellules injectées Complications infectieuses Absence complications infectieuses post-opératoires Évaluation fonctionnelle :

• Stade NYHA
• Fraction d'éjection
• Contractilité segmentaire

• III -> II
• 22 % -> 36%
• Augmentation (+)

Évolution viabilité segmentaire Amélioration (+) Nom du patient G. MYO 6 Age 55 Sexe M Type de Pathologie Insuffisance cardiaque post-ischémique Fraction d'éjection 34 % Stade classification NYHA IV PET-Scan Présence d'une zone non viable Échographie initiale Akinésie antérieure, akinésie latérale, dyskinésie apicale, hypokinésie septale Bilan virologique Négatif Durée de culture 16 J Cellules : nombre, caractéristiques 1210 x 10⁶. CD56+ : 85 % ; CD15+ : 10 % ; Desmine+ : 85 % ; HLA Classel : 96 % ; Viabilité : 97 %; Formation de myotubes (test fonctionnel) : +. Siège d'injection Antérieure ; 1150 x 10⁶ cellules injectées Complications infectieuses Avant injection, patient en état de choc cardiovasculaire, sous adrénaline et noradrénaline. Absence complications infectieuses post-opératoires (24 h) après injection. Évaluation fonctionnelle : Patient décédé le 28/04/01. Cause du décès non imputable à l'injection cellulaire (choc cardio-vasculaire suivi d'ischémie mésentérique probables)

• Stade NYHA
• Fraction d'éjection
• Contractilité segmentaire

Évolution viabilité segmentaire Sans objet The injection of autologous muscle cells, prepared according to the method, was again performed on 5 other patients. The following tables summarize clinical follow-up data for 4 of 5 patients (named MYO3, MYO4, MYO5 and MYO6, respectively). The clinical follow-up of the sixth patient is in progress. Patient's name D. MYO 3 Age 62 Sex M Type of Pathology Post-ischemic heart failure, unstable angina Ejection fraction 25% NYHA classification stage III PET Scan Presence of a non-viable area Initial ultrasound Anterior ankinesia, apical dyskinesia, lateral hypokinesia Virological assessment Negative Culture time 18 J Cells: number, characteristics 922 x 10 ⁶ . CD56 +: 91%; CD15 +: 6%; Desmin +: 65%; Viability: 98%; Formation of myotubes (functional test): +. Injection seat Anterior; 900 x 10 ⁶ cells injected Infectious complications Absence of postoperative infectious complications Functional assessment:

• NYHA Stadium
• Ejection fraction
• Segmental contractility

• III -> II
• 25% -> 35%
• Increase (+/-)

Evolution segmental viability Improvement (+) Patient's name E. MYO 4 Age 67 Sex M Type of Pathology Post-Ischemic Heart Failure Ejection fraction 31% NYHA classification stage III PET Scan Presence of a non-viable area Initial ultrasound Apical akinesia, posterior akinesis, lateral hypokinesia Virological assessment Negative Culture time 20 J Cells: number, characteristics 657 x 10 ⁶ . CD56 +: 97%; CD15 +: 15%; Desmin +: 58%; HLA Classel: 94%; Viability: 98%; Formation of myotubes (functional test): +. Injection seat Posterior; 620 x 10 ⁶ cells injected Infectious complications Absence of postoperative infectious complications Functional assessment:

• NYHA Stadium
• Ejection fraction
• Segmental contractility

• III -> II
• 31% -> 50%
• Increase (+/-)

Evolution segmental viability Improvement (+) Patient's name F. MYO 5 Age 39 Sex M Type of Pathology Post-Ischemic Heart Failure Ejection fraction 22% NYHA classification stage III PET Scan Presence of a non-viable area Initial ultrasound Apical akinesia, posterior akinesia, anterior hypokinesia, lateral hypokinesia Virological assessment Negative Culture time 14 J Cells: number, characteristics 993 x 10 ⁶ . CD56 +: 95%; CD15 +: 4%; Desmin +: 78%; HLA Classel: 96%; Viability: 98%; Formation of myotubes (functional test): +. Injection seat Postero-lateral; 950 x 10 ⁶ cells injected Infectious complications Absence of postoperative infectious complications Functional assessment:

• NYHA Stadium
• Ejection fraction
• Segmental contractility

• III -> II
• 22% -> 36%
• Increase (+)

Evolution segmental viability Improvement (+) Patient's name G. MYO 6 Age 55 Sex M Type of Pathology Post-Ischemic Heart Failure Ejection fraction 34% NYHA classification stage IV PET Scan Presence of a non-viable area Initial ultrasound Anterior ankinesia, lateral akinesia, apical dyskinesia, septal hypokinesia Virological assessment Negative Culture time 16 J Cells: number, characteristics 1210 x 10 ⁶ . CD56 +: 85%; CD15 +: 10%; Desmin +: 85%; HLA Classel: 96%; Viability: 97%; Formation of myotubes (functional test): +. Injection seat Anterior; 1150 x 10 ⁶ cells injected Infectious complications Before injection, patient with cardiovascular shock, adrenaline and norepinephrine. Absence of postoperative infectious complications (24 h) after injection. Functional assessment: Patient died on 28/04/01. Cause of death not attributable to cellular injection (cardiovascular shock followed by probable mesenteric ischemia)

• NYHA Stadium
• Ejection fraction
• Segmental contractility

Evolution segmental viability Not applicable

In summary, these tests showed the possibility of obtaining the desired number of cells within a limited time of 2 to 4 weeks. They have also shown that the collection, preparation and transplantation of human autologous muscle cells could be carried out according to the invention without difficulties and pre- or post-surgical complications.

Finally, these trials have also shown a clinical improvement, an increase in regional contractility (ultrasound) associated with an increase in the viable area area (PET-Scan) in these patients.

1. a) elles montrent que la transplantation des cellules d'origine musculaire améliore de manière significative la fonction ventriculaire gauche suite à un infarctus du myocarde,
2. b) que l'amélioration est clairement dépendante du nombre de cellules injectées,
3. c) que cette transplantation potentialise un traitement pharmacologique, notamment par une inhibition de l'ACE.
4. d) Et enfin que le procédé de l'invention est adapté pour le traitement de l'insuffisance cardiaque chez l'homme.

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In conclusion, the studies presented in parts B and C allow to bring the following different results:

1. a) they show that transplantation of muscle-derived cells significantly improves left ventricular function following myocardial infarction,
2. b) that the improvement is clearly dependent on the number of cells injected,
3. c) that this transplantation potentiates a pharmacological treatment, in particular by inhibition of ACE.
4. d) And finally that the method of the invention is suitable for the treatment of heart failure in humans.

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Claims (23)

1. A method for preparing a composition intended for cell therapy in humans, said method comprising the following steps:

   a) mincing a skeletal muscle tissue biopsy;

   b) enzymatic dissociation of the muscle fibres and cells and separating the individualized cells by filtering;

   c) culturing the cells obtained in step b) in a reactor for culturing adherent cells in the presence of a medium comprising MCDB 120 and D-valine substituted for L-valine until a population of cells comprising CD56+ cells as the dominant cell type is obtained, said cell type being dominant when the proportion of this cell type in said cell population exceeds 50%, said culture if necessary comprising one or more expansion phases;

   d) harvesting the cell population obtained in step c);

   e) if necessary, freezing the cell population harvested in step d).
2. A method according to claim 1, characterized in that the harvested cell population comprises 50 x 10⁶ cells to 800 x 10⁹ cells, preferably at least 500 x 10⁶ cells, among which myoblast cells expressing the CD56 marker constitute said dominant cell type.
3. A method according to claim 2, characterized in that the harvested cell population comprises at least 50%, preferably 60%, more preferably 70% of myoblast cells expressing the CD56 marker.
4. A method according to claim 2 or claim 3, characterized in that said medium contains a glucocorticoid and bFGF.
5. A method according to any one of claims 2 to 4, characterized in that the composition is intended for reconstituting skeletal, cardiac or visceral muscle tissue in humans.
6. A method according to any one of claims 2 to 5, characterized in that the composition is intended for the treatment of post-ischemic cardiac insufficiency in humans.
7. A method according to any one of claims 2 to 6, characterized in that the composition is intended for the treatment of innate or acquired muscular dystrophy.
8. A method according to any one of claims 2 to 7, characterized in that said mincing step is assisted by mechanical or electrical blades.
9. A method according to any one of claims 2 to 8, characterized in that it further comprises a step for CD34+ cell depletion.
10. A cell therapy product for its use in the treatment of a cardiac pathology in humans, comprising a population of 500 x 10⁶ to 800 x 10⁹ cells, said population comprising at least 50% of CD56+ cells.
11. A cell therapy product according to claim 10, characterized in that said population comprises at least 60% CD56+ cells.
12. A cell therapy product according to claim 10, characterized in that said population comprises at least 70% CD56+ cells.
13. A cell therapy product according to any one of claims 10 to 12, characterized in that said treatment of a cardiac pathology is a treatment by repair of cardiac tissue in humans.
14. A cell therapy product according to any one of claims 10 to 13, characterized in that said cardiac pathology is post-ischemic cardiac insufficiency.
15. A cell therapy product according to claim 14, characterized in that said post-ischemic cardiac insufficiency treatment is carried out concomitantly with a treatment by an angiotensin conversion enzyme inhibitor (ACEI).
16. A cell therapy product according to any one of claims 10 to 15, characterized in that said treatment of a cardiac pathology is a treatment by autologous transplantation.
17. Use of a cell population as defined in any one of claims 10 to 16, for the production of a cell therapy product intended for the treatment of a cardiac pathology.
18. A culture medium comprising MCDB 120 and D-valine substituted for L-valine.
19. A culture medium according to claim 18, which does not comprise phenol red and which does not comprise thymidine.
20. In vitro use of a culture medium according to claim 18 or claim 19, for the differentiation of skeletal muscle tissue cells into myoblasts.
21. In vitro use of a culture medium according to claim 18 or claim 19, for the production of a cell population comprising a dominant cell type selected from CD34+ cells, CD15+ cells, CD56+ cells, HLA class 1 cells, said cell type being dominant when the proportion of this cell type exceeds 50%.
22. Use according to claim 21, characterized in that said dominant cell type is selected from CD34+ cells, CD15+ cells and CD56+ cells.
23. In vitro use of a culture medium according to claim 18 or claim 19, for the production of a cell therapy product comprising a cell population comprising CD56+ cells as the dominant cell type, said cell type being dominant when the proportion of said cell type in said population exceeds 50%.

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